The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation.

Abstract:

The gene 4 protein of bacteriophage T7, a functional hexamer, comprises DNA helicase and primase activities. Both activities depend on the unidirectional movement of the protein along single-stranded DNA in a reaction coupled to the hydrolysis of dTTP. We have characterized dTTPase activity and hexamer formation for the full-length gene 4 protein (gp4) as well as for three carboxyl-terminal fragments starting at residues 219 (gp4-C219), 241 (gp4-C241), and 272 (gp4-C272). The region between residues 242 and 271, residing between the primase and helicase domains, is critical for oligomerization of the gene 4 protein. A functional TPase active site is dependent on oligomerization. During native gel electrophoresis, gp4, gp4-C219, and gp4-C241 migrate as oligomers, whereas gp4-C272 is monomeric. The steady-state k(cat) for dTTPase activity of gp4-C272 increases sharply with protein concentration, indicating that it forms oligomers only at high concentrations. gp4-C219 and gp4-C241 both form a stable complex with gp4, whereas gp4-C272 interacts only weakly with gp4. Measurements of surface plasmon resonance indicate that a monomer of T7 DNA polymerase binds to a dimer of gp4, gp4-C219, or gp4-C241 but to a monomer of gp4-C272. Like the homologous RecA and F(1)-ATPase proteins, the oligomerization domain of the gene 4 protein is adjacent to the amino terminus of the NTP-binding domain.

Polymerases:

T7

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